Project Summary/Abstract. Tobacco addiction is the second leading cause of death in the world and the single largest cause of cancer and heart disease. Currently there are 1.3 billion tobacco smokers worldwide resulting in an estimated 6 million deaths a year, and given present smoking trends, tobacco will kill 10 million people each year by 2020. Each year nearly half of the 42 million adult smokers attempt to quit; yet due to the highly addictive nature of nicotine, less than 5% succeed. Aids to smoking cessation include supportive counseling, nicotine replacement and receptor antagonists that reduce nicotine reward and withdrawal symptoms. Unfortunately, long-term outcomes for nicotine replacement therapies remain poor and achieve an abstinence rate of only 10-20% after the first year. Drug of abuse vaccines induce antibodies that block the pharmacological effects of drugs like nicotine. To date, vaccines for smoking cessation have shown promise in preclinical animal models for their ability to diminish nicotine-mediated physiological and behavioral responses. However, in clinical studies these vaccines failed to measure significant differences in smoking abstinence between the intervention and placebo groups. The Achilles heel with nicotine vaccines has been their inability to generate high enough titers (antibody concentration) to reduce free drug. In view of the limitations with immunotherapy, how can sufficient pharmacokinetic (PK) capacity be engendered to prevent rapid nicotine distribution into the brain after smoked drug intake? We came to envision that to overcome such forbidding needs of extremely high nicotine-antibody titers would require a biologic able to catabolize nicotine rather than simply sequestering the drug. The proposal at hand details a bacterial strain, Pseudomonas putida, which has evolved to use nicotine as its sole source of carbon and nitrogen. From this bacterial strain we disclose a first in class enzyme, a nicotine oxidoreductase (NicA2) that is highly efficient at degrading nicotine to a non-psychoactive product. Our initial characterization of the enzyme indicates it to be an excellent candidate for what we term ?enzympharmacotherapy?. Yet, successful demonstration of such a strategy as a smoking cessation therapy will require mastery of a number of challenges that we will undertake as specific aims including: (1) Biochemical studies as a means to unravel NicA2's mechanism of action and specificity. (2) Engineering NicA2 to increase its stability in serum and reduce immunogenicity liabilities. (3) Evaluation of the efficacy of NicA2 and variants in rats using blood/brain distribution, and behavioral models. Our research initiative is high-risk, however, if successful, enzympharmacotherapy will address the shortcomings of antibody-based therapeutics through a new biologic capable of catalyzing nicotine's degradation.